Composition containing fine particles for supporting biologically active substance thereon or having the same supported thereon and method for preparing these

ABSTRACT

Provided are a dispersion comprising organic-inorganic hybrid type particles carried thereon with a biologically active substance, wherein the above particles can be obtained by allowing a block copolymer represented by Formula (I):
 
PEG-block-poly(carbo)  (I)
 
(wherein PEG represents a polyethylene glycol segment, and carbo represents a repetitive unit having a carboxylate ion on a side chain) and an aqueous medium system capable of forming hydroxyapatite to coexist with the biologically active substance, and a preparing method for the same.

TECHNICAL FIELD

The present invention relates to a composition containingorganic-inorganic hybrid particles for carrying a biologically activesubstance or carried thereon with it and a method for preparing them.The above biologically active substance can preferably be a highmolecular polyvalent anionically chargeable compound (for example, poly-or oligonucleotide and poly- or oligopeptide).

BACKGROUND ART

A crystal of calcium phosphate (hydroxyapatite) formed when a calciumaqueous solution is mixed with a phosphoric acid aqueous solution sothat a supersaturation state is obtained has a property to bond withDNA. A method in which calcium phosphate and DNA are coprecipitatedmaking use of this property has been widely utilized as a method forintroducing DNA into a cell. The problems of this method include a verynarrow range of an optimum condition, which makes handling thereofdifficult, and less liability to obtain reproducibility. In particular,growth in the crystal of calcium phosphate is very fast to form thegiant crystal, and therefore it is pointed out that an efficiency ofintroducing DNA is reduced if a calcium aqueous solution and aphosphoric acid aqueous solution are not quickly worked on a cell aftermixing. Also, hydroxyapatite is used as a base substance (for example, acarrier for chromatography) for adsorbing a biologically activesubstance including polynucleotide such as DNA, other peptides orpolypeptide.

In the foregoing method for introducing DNA (or gene) into a cell,development of a method for controlling a growth in a crystal of calciumphosphate and a particle diameter thereof is considered to be importantto a rise in an introducing efficiency of DNA, a reproducibility and astorage stability. Further, if substances carried or adsorbed on suchcrystal can widely be used as various biologically active substanceswithout being restricted to DNA described above and a particle diameterof a particle formed can be controlled, it is considered to be importantin providing a carrier system which can widely be used for carryingmedicines or a delivery system for medicines. Accordingly, an object ofthe present invention is to provide a composition useful for formingparticles which not only enhance an efficiency of introducing DNA into acell but also have a wide and controlled particle diameter and which canstably carry a biologically active substance and conveniently deliverthe biologically active substance to a target cell, a desired tissue ora local site.

DISCLOSURE OF THE INVENTION

The present inventors have continued researches in order to control agrowth in a crystal of calcium phosphate (mainly hydroxyapatite) in anaqueous solution containing a calcium ion and a phosphoric acid ion anda particle diameter thereof. As a result thereof, they have found thatcalcium phosphate particles into which DNA or the other biologicallyactive substances is introduced or in which they coexist can be formedwhile controlling a particle diameter thereof when a calcium ion isreacted with a phosphoric acid ion under the coexistence of DNA or theother biologically active substances in an aqueous solution in whichpresent is a specific block copolymer containing a hydrophilic andnonionic polyethylene glycol (PEG) segment and a polyanionic segmentoriginating in a carboxyl group. In addition thereto, it has beenconfirmed that a particle diameter of such particles can be controlled,if necessary, to a submicron order (several 100 nm) or less and that anaqueous dispersion system containing such particles can stably be storedunder an ambient condition without producing precipitates. Further, ithas been found as well that such aqueous dispersion system can be turnedinto a composition of a dried type (for example, freeze-dried) and thatit can be then reconstituted to the same aqueous dispersion system.

Hence, according to the present invention, provided is an aqueouscomposition for forming (or used for forming) organic-inorganic hybridtype particles carried thereon with a biologically active substance,wherein the particles described above comprise a block copolymer havinga structure represented by Formula (I):PEG-block-poly(carbo)  (I)(wherein PEG represents a polyethylene glycol segment, and carborepresents a repetitive unit having a carboxylate ion on a side chain),a calcium ion (Ca⁺²) and a phosphoric acid ion (PO₄ ³⁻) as essentialcomponents.

Further, provided as another embodiment of the present invention is acomposition comprising organic-inorganic hybrid type particles carriedthereon with a biologically active substance, wherein the particlesdescribed above are formed from a block copolymer having a structurerepresented by Formula (I):PEG-block-poly(carbo)  (I)(wherein PEG represents a polyethylene glycol segment, and carborepresents a repetitive unit having a carboxylate ion at a side chain),a calcium ion (Ca⁺²), a phosphoric acid ion (PO₄ ³⁻) and the abovebiologically active substance, and the above particles have an averageparticle diameter of 50 to 60 nm.

Further, provided are an aqueous dispersion composition for formingorganic-inorganic hybrid type particles carried thereon with suchbiologically active substance, a method for preparing a compositioncontaining the above particles carried thereon with a biologicallyactive substance and a method for introducing the above biologicallyactive substance, particularly poly- or oligonucleotide into a cell,comprising a step of incubating the above composition under thecoexistence of a cultured cell or injecting it into a suitable part ofan animal.

It has so far been known that when a phosphoric acid ion is added to acoagulating solution of modified PEO-block-PMAA (PEO is a polyethyleneoxide or polyethylene glycol segment; PMAA is a polymethacrylic acidsegment; and among PMAA, three segments are modified with C₁₂-alkane)after adding CaCl₂, a hybrid type structure ofhydroxyapatite/PEG-block-PMAA-C₁₂ having a neuronal structure isobtained (Helmut Cölfen, Macromol. Rapid Commun. 2001, 22, 219 to 252).

According to the present invention, provided are particles which aresubstantially spherical regardless of that a biologically activesubstance is further carried or absent and a means capable of forminguniform particles which have a particle diameter suited for beingefficiently introduced into a cell by endocytosis and in which aparticle diameter has a narrow distribution. Particles in which abiologically active substance is absent shall be useful as ahydroxyapatite material of a new form comprising fine particles.Further, a system containing particles carried thereon with abiologically active substance shall deliver the biologically activesubstance to a target and expand a range of a usefulness in the abovesubstance.

BRIEF DESCRIPTION Of The DRAWINGS

FIG. 1 is a graph showing an influence of PAA and PEG-PAA exerted to agrowth in crystal of CaP, wherein (a) is a graph showing a change in atransmittance with the passage of time in the presence of PAA (PAAconcentration: (□) 14 μg/mL, (◯) 43 μg/mL and (Δ) 57 μg/mL), and (b) isa transmittance after 3 minutes since mixing the solutions ((□) PAA, (◯)PEG/PAA (blended) and (Δ) PEG-PAA).

FIG. 2 is a graph showing a result of measuring a particle diameter ofCaP particles by dynamic light scattering.

FIG. 3 is a graph showing a change in a polydispersity of CaP particlesto a PEG-PAA concentration.

FIG. 4 is a graph showing the same measuring result as in FIGS. 2 and 3regarding particles prepared without adding DNA in preparing theparticles.

FIG. 5 is an elution pattern showing a result of determining DNAincluded in CaP particles by HPLC; (a) upper line: only DNA and (b) agraph showing a change in an amount of included DNA to a PEG-PAAconcentration ((◯): eluate 1 and (Δ): eluate 2).

In FIG. 6, A) is a microscopic image in place of a drawing showing thestate of a cell after working only DNA on the cell. In this case,granular fluorescence indicating introduction by endocytosis isobserved.

B) is a microscopic image in place of a drawing showing the state of acell after working a precipitate (PEG-PAA 70 μg/ml) of CaP on the cell.In this case, an image showing that the precipitate is adsorbed on thesurface of the cell is obtained.

In FIG. 7, C) is a microscopic image in place of a drawing showing thestate of a cell after working CaP particles (PEG-PAA 280 μg/ml) (in thenon-coexistence of PMA) on the cell. In this case, granular fluorescenceindicating introduction of the CaP particles by endocytosis is observed.The same result was obtained in a PEG-PAA of 140 μg/ml.

D) is a microscopic image in place of a drawing showing the state of acell after working CaP particles (PEG-PAA 280 μg/ml) (in the coexistenceof PMA) on the cell. In this case, observed is a fluorescent imageshowing localization of the CaP particles in a nucleus as well asgranular fluorescence indicating that the CaP particles are introducedby endocytosis.

In FIG. 8, E) is a microscopic image in place of a drawing showing thestate of a cell after working only a fluorescent molecule Rhodamine onthe cell. In this case, fluorescence is observed all over a cytoplasma,and it is shown that a fluorescent molecule does not selectively move toa nucleus.

FIG. 9( a) is a graph showing that a particle diameter of theorganic-inorganic hybrid particles according to the present inventionchanges by a change in a copolymer concentration and a phosphoric acidconcentration in Example 9, and (b) is a graph showing a polydispersityof the respective particles.

FIG. 10 is a graph showing a dependency of a DNA incorporated amount inthe particles on copolymer and phosphoric acid concentrations.

FIG. 11( a), (b) and (c) are graphs showing results obtained byevaluating introduction of DNA into a cell in Example 8.

FIG. 12 is a graph showing a result of a test carried out in order toevaluate a toxicity of the particles prepared in Example 9.

FIGS. 13A and B are graphs showing results obtained by investigating anexpression activity of a plasmid DNA-incorporating particle in a cell inExample 10.

BEST MODE FOR CARRYING OUT THE INVENTION

According to the present invention, a block copolymer having a structurerepresented by Formula (I):PEG-block-poly(carbo)  (I)(wherein PEG represents a polyethylene glycol segment, and carborepresents a repetitive unit having a carboxylate ion on a side chain)is characterized by being used in common in a system forming calciumphosphate (hydroxyapatite).

As described above, when referred to as “calcium phosphate” or“hydroxyapatite” in the present specification, it means a mixture ofsalts which contains mainly calcium phosphate (Ca₃(PO₄)₂) andhydroxyapatite (Ca₁₀(OH)₂(PO₄)₆) and which is formed from a calciumcation (Ca⁺²) and a phosphoric acid anion (PO₄ ³⁻) in an aqueoussolution. It is intended that 50% by weight or more of a salt of ahydroxyapatite type is preferably contained therein.

Poly(carbo) which is one segment in the block copolymer described aboverepresents a polymer segment comprising a repetitive unit having acarboxylate ion at a side chain, and the kind of a starting materialproviding such repetitive unit does not matter as long as it meets theobjects of the present invention. However, capable of being preferablygiven is a repetitive unit originating in a compound having at least onecarboxyl group selected from the group consisting of aspartic acid,glutamic acid, methacrylic acid, acrylic acid and N-acetylhyalobiuronicacid (a repetitive unit of hyaluronic acid). In such Poly(carbo), afixed carboxyl group can stay in the form of an ester (for example,lower alkyl having up to 6 carbon atoms or benzyl ester) according to aproduction method of the block copolymer described above. According tothe present invention, a residue in the ester of such form may becontained in an amount of up to about 50%, preferably less than 10% andparticularly preferably 0% as long as introduction into or adsorptiononto calcium phosphate (or hydroxyapatite) is not hindered.

The term “having the structure” represented by Formula (I) intends thata linkage group between PEG and poly(carbo) and an end of PEG orpoly(carbo) can have any group or part as long as it meets the objectsof the present invention.

A copolymer represented by any one of the following Formulas (II-a),(II-b), (III-a) and (III-b) can be given as the block copolymerparticularly preferably used in the present invention:

In the respective formulas, the respective codes each have independentmeanings;

A represents a hydrogen atom or a substituted or unsubstituted alkylgroup having up to 12 carbon atoms;

L represents a single bond, NH, CO or X(CH₂)_(p)Y, in which X representsOCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO; Y represents NH or CO; andp represents an integer of 1 to 6;

T represents a hydrogen atom, a hydroxyl group or —ZR, in which Zrepresents a single bond, CO, O or NH, and R represents a substituted orunsubstituted hydrocarbon group having up to 12 carbon atoms;

m represents an integer of 4 to 2500; and

x+y or z represents an integer of 5 to 300, provided that a carboxylateion present can form a carboxyester residue in an amount of up to 50%.Also, the mark “·” between an α-aspartic acid unit and a β-aspartic acidunit in Formulas (II-a) and (II-b) described above means that theseunits are present at random.

The block copolymer in which m is an integer of 12 to 2500 in theformulas described above and in which x+y or z is an integer of 5 to 50can more preferably be used.

The definitions of the respective groups and the respective parts in theformulas described above have, to be specific, the following meanings.The “alkyl group having up to 12 carbon atoms (hereinafter abbreviatedas C₁₂, and such describing manner shall be applied as well whenrepresenting the other groups having carbon atoms)” is an alkyl groupwhich may be linear or branched and represents, for example, methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, n-pentyl, n-hexyl andn- or iso-dodecyl. A substituent for such alkyl groups may be any groupas long as it meets the objects of the present invention, and capable ofbeing preferably given are a hydroxyl group, a carboxyl group, a grouprepresented by a formula R¹R²CH— (wherein R¹ and R² representindependently C₁₋₁₀ alkyloxy, aryloxy or aryl-C₁₋₃ alkyloxy or representethylenedioxy (—O—CH(R′)—CH—O—, wherein R′ is a hydrogen atom or a C₁₋₆alkyl group) which may be substituted with C₁₋₁₀ alkyl) and a grouprepresented by a formula R¹′ R²′ NCH₂— (wherein R¹′ and R²′ representindependently an amino protective group of an organic silyl type, forexample, a trialkylsilyl group, or R¹′ and R²′ represent an atomic groupwhich can form a 4- to 7-membered disilaneazacyclo heterocyclic ringtogether with a nitrogen atom to which they are bonded). For example,the group represented by the formula R¹R² CH— represents so-calledacetal or ketal part and can readily be converted to OCH— (aldehydegroup) by moderate hydrolysis. On the other hand, the group representedby the formula R¹′ R²′ NCH₂— can readily be converted to H₂N— in asolution containing, for example, tetraalkylammonium chloride.Accordingly, the block copolymer represented by Formula (II-a), (II-b),(III-a) or (III-b) having such substituent is used to form theorganic-inorganic hybrid type particles (for example, polymer micelle)according to the present invention, and then the foregoing substituentwhich is usually present on the shell or the surface of the aboveparticles is converted to an aldehyde group or an amino group; andpolypeptide showing a specific bonding property in, for example, anantibody is advantageously subjected to covalent bonding with the aboveparticles via the functional group thus obtained. Thus, the aboveparticles can be provided with a target directivity. A method forgetting a PEG segment having such substituent is known and can bereferred to WO96/33233 (or corresponding U.S. Pat. No. 5,925,720) in thecase of, for example, the R¹R²CH— group.

The block copolymer described above can be selected from conventionallyknown ones. It can be produced by forming first a polyethylene glycolsegment and then forming a poly(amino acid) segment according to, forexample, a method described in Japanese Patent Application Laid-Open No.107565/1994 or by forming first a polyethylene glycol segment and thenforming a poly((meth)acrylic acid) segment according to a methoddescribed in WO97/06202. As another method, a conventionally knownlinkage group may be present, if necessary, between a PEG segment and apoly(carbo) segment. However, the copolymer described above shall not berestricted to those obtained by these production methods, and copolymerswhich can be obtained by producing in advance independently polymersconstituting both segments and then combining them by a suitable methodcan be used as well in the present invention.

In the block copolymer thus obtained, T in Formulas (II-a), (II-b),(III-a) and (III-b) described above or an end group correspondingthereto is usually a hydrogen atom or a hydroxyl group, and a —ZR groupcan be introduced into these end groups by a conventionally knownmethod. Such R shall not be restricted, and hydrocarbon groups such as—CH₃, —CH₂CH₃, —CH═CH₂,

can be given. Such groups can be introduced according to the methoddescribed in U.S. Pat. No. 5,925,720 described above.

The biologically active substance which is intended to be or is carriedon (or included in) the organic-inorganic hybrid type particlesaccording to the present invention may be any biologically activesubstance, though shall not theoretically restricted, as long as it isan organic compound which can be included in or adsorbed on a complex ora cross-linked matrix formed by a block copolymer (particularly acarboxylate ion) and a calcium ion or hydroxyapatite. Preferably,however, it may be any biologically active substance selected from thegroup consisting of poly- or oligonucleotide (used in a conceptincluding DNA or RNA or peptide derivatives thereof) and poly oroligopeptide or derivatives thereof. Such DNA can be a conventionallyknown cancer-inhibiting gene and others, a gene necessary formaintaining a homemostasis of organisms and an anti-sense of the otherspecific genes. According to the present invention, these genes canefficiently be introduced into a target cell. Further, in order to usein combination with the organic-inorganic hybrid type particles carriedthereon with such genes or independently, polypeptide (including singleprotein and protein having a sugar chain) which is a proliferativefactor known to accelerate a differentiation and inhibit a proliferationin a cell and induce apotosis can be selected as a biologically activesubstance carried on the above particles. For example, TGF-β₁, TGF-β₂,TGF-α, connective tissue-activating peptide, a tumor necrotizing factor,an insulin-like proliferative factor, interleukin, a colony stimulatingfactor and a nerve proliferating factor can be given as thesebiologically active substances.

The factor effective for healing of wound is included in theseproliferative factors, and the organic-inorganic hybrid type particlesof then present invention carried thereon with them can be applied to awound part and used for accelerating healing of wound. Further, if ablood coagulating factor, for example, thrombin is carried as abiologically active substance on the above particles in relation tohealing of wound or regardless thereof, it can be used for acceleratinghemostatis in combination with a calcium ion or without combiningtherewith. If the particles carrying (or including) such thrombin areapplied to a wound part in a dry state or a concentrated suspensionstate, it works on fibrinogen present in bleeding blood to form fibrin,which shall bring about so-called hemostatis in situ. Accordingly, acomposition containing such particles is useful as a composition forhemostatis.

An antiviral agent, an antibacterial agent, an antihistaminic agent, anantitumor agent and a bone inducing agent may be carried as the otherbiologically active substance on the above particles according to thepresent invention as long as they can be carried on the above particles.

According to the present invention, provided is an aqueous dispersioncomposition for forming the organic-inorganic hybrid type particlescarried thereon with the biologically active substance comprising suchblock copolymer, a calcium ion and a phosphoric acid ion as essentialcomponents. The “aqueous dispersion composition” referred to herein oran “aqueous dispersion” referred to later means a solution, a dispersionand a suspension comprising a solvent system which comprises water as aprincipal solvent and which may contain, if necessary, a small amount ofa water-miscible organic solvent (for example, methanol, ethanol andacetone) as long as it does not exert an adverse effect in achieving theobjects of the present invention. A buffer which can control the pH to6.8 to 7.8 is preferably contained in these solutions. A calcium ion anda phosphoric acid ion each contained in these solutions can originate inthe respective corresponding water-soluble salts. Typically, the formeris derived from calcium chloride, and the latter is derived fromdisodium hydrogenphosphate.

A content proportion of a calcium ion and a phosphoric acid ion isconsiderably important in the present invention, and a calcium ion hasto be present in an amount which is excessive as compared with anequivalent required for reacting both to form hydroxyapatite(Ca₁₀(OH)₂(PO₄)₆). To be specific, a proportion of Ca⁺² to PO₄ ³⁻ can be50 to 200:1 in terms of a mole concentration. When a calcium ion and aphosphoric acid ion are present in such proportion, the block copolymerdescribed above suitably interacts with calcium phosphate(hydroxyapatite) and is bonded, cross-linked or adsorbed.

Further, Ca⁺² can be present, though shall not be restricted, in anamount of 60 to 300 mM, and PO₄ ³⁻ can be present, though shall not berestricted, in an amount of 0.4 to 10 mM in the aqueous compositiondescribed above. Such proportions are suited for providing the aqueousdispersion comprising the organic-inorganic hybrid type particlescarrying or including such biologically active substance, including theaqueous composition described above containing the biologically activesubstance according to the present invention. To be specific, the blockcopolymer and the biologically active substance described above suitablyinteract with calcium phosphate (hydroxyapatite) and are bonded oradsorbed. For example, “the particles carried thereon with thebiologically active substance” referred to in the present inventionmeans particles staying in a state in which a part or the whole of thebiologically active substance is included in the inside of the particlesor in which a part or the whole thereof is present on the surface of theparticles.

On the other hand, the block copolymer can be present, though shall notbe restricted, in an amount of 10 to 500 μg/ml in the aqueouscomposition described above. The foregoing concentrations of Ca⁺² andPO₄ ³⁻ in the aqueous composition described above and the concentrationof the block copolymer described immediately before are usually suitedfor stably dispersing the organic-inorganic hybrid type particles(containing no biologically active substance) formed in the aboveaqueous composition or the organic-inorganic hybrid type particles(containing the biologically active substance) in the aqueous dispersionin an aqueous solution. However, the aqueous composition or the aqueousdispersion which meets the objects of the present invention can beprovided even if the respective components are used in concentrationsexceeding the concentrations described above. Also, such aqueousdispersion can be converted to a dry form by a conventional method, forexample, a dry freezing method. The composition of such dry form can beconstituted again to a stable aqueous dispersion by adding an aqueousmedium. Further, it can be turned, if necessary, into a formulation ofanother form using another binders as it stays in the dry form.

The organic-inorganic hybrid type particles described above (containingno biologically active substance) can be used for forming fine ormicroscopic hydroxyapatite of a submicron order having a uniformparticle diameter. Such particles can be obtained, if necessary, byremoving excess Ca⁺² by dialysis and then freeze-drying.

The aqueous dispersion according to the present invention which hasalready been partially referred to in the above can be prepared byallowing the biologically active substance to coexist in the aqueouscomposition described above. To be more specific, it can be prepared by,though shall not be restricted, (A) preparing a first aqueous solutioncontaining a biologically active substance, a calcium ion and, ifnecessary, a buffer, (B) preparing independently a second aqueoussolution containing the block copolymer having the structure representedby Formula (I):PEG-block-poly(carbo)  (I)(wherein PEG represents a polyethylene glycol segment, and carborepresents a repetitive unit having a carboxylate ion at a side chain),a phosphoric acid ion and, if necessary, a buffer and(C) mixing the first aqueous solution described above with the secondaqueous solution on a condition enough for forming hydroxyapatite. Asalt such as sodium chloride can be contained in the second aqueoussolution, and when the buffer is used, it is preferably selected so thata pH of the final dispersion can be controlled to 6.8 to 7.8.

Though shall not be restricted to such production method, the particlescontained in the aqueous suspension according to the present inventionare particles carried thereon with the biologically active substancewhich are formed from the block copolymer described above, a calciumion, a phosphoric acid ion and the biologically active substance andhave an average particle diameter of 50 to 600 nm. According to theforegoing production method of the present invention, an aqueousdispersion containing very uniform particles in which an averageparticle diameter has any size of 50 to 600 nm and in which apolydispersity is 0.1 or less can be provided by selecting aconcentration of the block copolymer. It is a matter of course thatparticles having a particle diameter of a several μm order exceeding 600nm can be formed, if necessary, by extending preparing time. Theseaqueous dispersions can be stored for several days to one month on anambient condition (for example, room temperature) without substantiallycausing precipitation or phase separation, and therefore they can beused as a composition for injection as they are or, if necessary, byremoving excess ionic low molecular compounds by dialysis orultrafiltration.

The biologically active substance which can be carried on such particlesis, though described above, generally a compound which can show anyuseful activity in organisms of animals (particularly human beings) andcan be polynucleotide (including DNA, mRNA and the like) coding exoticactive peptide, polynucleotide coding a function which promotes orcontrols revelation of a specific gene, polynucleotide such asanti-sense DNA and ribozime and polypeptide (including protein and, aslong as the objects are met, oligopeptide). These peptides preferablycontain a polyvalent carboxyl group.

The organic-inorganic hybrid type particles according to the presentinvention comprise typically, though shall not be restricted,

30 to 70% by weight of the block copolymer,

25 to 65% by weight of hydroxyapatite and

0.1 (preferably 1) to 15% by weight of the biologically active substanceeach based on the whole weight of the above particles.

The production method for the aqueous dispersion according to thepresent invention can be provided by carrying out the specificembodiment in the presence of a cultured cell, for example, as a methodfor introducing polynucleotide into an animal cell, characterized by:

(A) adding an aqueous dispersion comprising organic-inorganic hybridtype particles carried thereon with a biologically active substance to acultured substance of an animal cell, wherein the above particles areformed from a block copolymer having a structure represented by Formula(I):PEG-block-poly(carbo)  (I)(wherein PEG represents a polyethylene glycol segment, and carborepresents a repetitive unit having a carboxylate ion on a side chain),a calcium ion (Ca⁺²), a phosphoric acid ion (PO₄ ³⁻) and the abovebiologically active substance; the above particles have an averageparticle diameter of 50 to 600 nm; and the above biologically activesubstance is selected from the group consisting of poly- oroligonucleotide and poly- or oligopeptide and(B) incubating the cultured substance prepared in (A).

According to such method, the particles can slowly be dissolved under aphysiologic condition by reducing a concentration of the block copolymerin forming the particles described above, and on the other hand, theparticles can stably be maintained under the physiologic condition byelevating a concentration of the block copolymer, which makes itpossible as well to control a discharge time of the biologically activesubstance in a target part. For example, DNA as the biologically activesubstance is introduced into a cell in the form of the organic-inorganichybrid type particles described above, and it can be delivered to thenucleus of the cell while avoiding decomposition caused by nuclease inthe cell. Such a high efficiency of introducing DNA into a cell can beachieved as well when the composition of the present invention isinjected into the suited part of an organism, that is, in a system invivo.

As shown above, according to the present invention, the aqueousdispersion comprising the organic-inorganic hybrid type particles whichcan be a carrier for a biologically active substance or the compositionof a dry form can be provided, or the aqueous composition used forproviding the same and the preparing method for them can be provided.

The present invention shall more specifically be explained below whilegiving the examples of the specific embodiments of the present inventionin order to simplify the explanations, but the present invention shallnot be intended to be restricted to them.

Example 1 Experiment for Confirming Action and Effect of Block Copolymer

This experiment shows that a specific block copolymer is effective foran inhibition in the formation of precipitates of calcium phosphate(hereinafter abbreviated as CaP) or a controlled formation in particleshaving a fixed particle diameter or less.

<Experimental Method>

(1) The following aqueous solution was prepared:

Solution A: DNA (16 mer: 70 μg/mL) 1/10 TE buffer (pH 7.6) Ca⁺² 250 mM(using CaCl₂) Solution B: PO₄ ³⁻ 1.5 mM (using Na₂HPO₄) Hepes buffer 50mM (pH 7.05) NaCl 140 mM poly(aspartic acid) homopolymer (hereinafterabbreviated as PAA) or PEG- block-poly (aspartic acid) (hereinafterabbreviated as PEG-PAA; PEG molecular weight: 12000, PAA polymerizationdegree: 24)

In the above, PAA is poly(α,β)-DL-aspartic acid, and a compound having amolecular weight of 2000 to 10000 (polymerization degree: 15 to 77)(obtained from SIGMA) is used. PEG-PAA is represented by the followingformula:CH₃O(CH₂CH₂O)_(n)—(COCH(CH₂COO⁻)NH)_(x).(COCH₂CH(COO⁻)NH)_(y)Hand a compound in which a PEG segment had a molecular weight of about12000 and in which a PAA segment had a polymerization degree (x+y) of 24was produced and used.(2) The solution A was mixed with the solution B at 37° C. to trace achange in a turbidity from a transmission factor of light having awavelength of 350 nm.<Result>

The result is shown in FIG. 1. When a homopolymer of PAA was added, thetransmission factor was suddenly reduced immediately after mixing thesolutions. The degree thereof was dependent on a PAA concentration, andthe higher the polymer concentration was, the more largely thetransmission factor was changed (FIG. 1 a). In this case, if PEGcoexisted, the transmission factor was not changed, and it was suggestedthat PEG did not interact with precipitate (FIG. 1 b). On the otherhand, it was shown that the transmission factor was scarcely changedunder the presence of the block copolymer and that precipitate wasinhibited from being formed. It is apparent from these results that thecopolymer structure is necessary for inhibiting precipitation ofDNA-including CaP.

Example 2 Particle Diameter of Composite Particles of DNA and CalciumPhosphate (No. 1)

The solution A was mixed with the solution B in the same manner as inExample 1 to prepare an aqueous dispersion containing compositeparticles. The dispersion was left standing still at 37° C. for a nightafter mixing, and then the particle diameter was evaluated by dynamiclight scattering (DLS) measurement of the dispersion.

DSL-7000 manufactured by Ohtsuka Electron Co., Ltd. was used as themeasuring apparatus. A light of argon laser having a wavelength of 488nm was used as an incident light to carry out the measurement at 25° C.A scattered light at an angle 90° to the incident light was detected toanalyze a time dependency in an intensity change thereof by a cumulantmethod, whereby a diffusion coefficient of the particles was determined.The diffusion coefficient thus obtained was converted to the particlediameter according to the following equation of Stokes-Einstein:R=kT/(6πηD)wherein R=particle diameter, k=Boltzmann's constant, η=viscositycoefficient, D=diffusion coefficient<Result>

The result of measuring the CaP particles by dynamic light scattering isshown in FIG. 2. It was confirmed by DLS measurement that the particleswere formed. The particle diameter thereof was 125 nm at a PEG-PAAconcentration of 70 μg/ml and decreased as the copolymer concentrationwas increased, and it was about 90 nm at 140 μg/ml. Then, the particlediameter was elevated again as the copolymer concentration wasincreased.

Further, the polydispersity which was an index of a size in a particlediameter distribution of the particles was determined similarly by thecumulant method to find that it was 0.1 or less. This value is adeviation of a standardized diffusion coefficient, and when it is 0.1 orless, it is usually regarded as monodispersibility in a colloidalparticle. Refer to FIG. 3.

Example 3 Particle Diameter of Composite Particles of Calcium PhosphateContaining No DNA

The same procedure as in Example 2 was repeated, except that a solutionobtained by removing DNA from the solution A prepared in Example 1 wasused in producing composite particles. The result thereof is shown inFIG. 4. The particle diameter and the polydispersity are almost the sameas in Example 2.

Example 4 Determination of Amount of DNA Introduced (or Included) intoParticles (No. 1)

CaP particles were prepared on the same condition as in Example 2. Theaqueous dispersion thus prepared was subjected to high performanceliquid chromatography (HPLC) on the following conditions toquantitatively determine DNA.

HPLC condition: column Superose 6HR (room temperature)

-   Eluent 1 CaCl₂ 125 mM, 140 mM NaCl, 50 mM HEPES, pH 7.4-   Eluent 2 CaCl₂ 200 mg/L (calcium ion 1.8 mM), NaH₂PO₄.H₂O 125 mg/L    (phosphoric acid ion 0.9 mM) NaCl 6400 mg/L, HEPES 5958 mg/L, pH 7.4-   Detection UV 260 nm    <Result>

The measuring result of HPLC is shown in FIG. 5. In the case of DNAalone, a peak originating in DNA was observed in the vicinity of anelution time of 30 minutes. On the other hand, in the case of the CaPsolution, a peak originating in the particles was confirmed togetherwith a peak originating in DNA in the vicinity of an elution time of 12minutes. A proportion of DNA included in the particles was calculatedfrom comparison of a ratio of both.

Under the conditions of the eluent 1, DNA was decreased from anincluding amount of 45% in a PEG-PAA concentration of 70 μg/mL as theconcentration was increased.

In the case where the eluent 2 which was close to a physiologiccondition was used, it was suggested that the particles were slowlydissolved at a low polymer concentration. On the other hand, it wassuggested that the particles were stable at a high polymer concentrationeven on a physiologic condition.

A composition weight ratio of the CaP particles formed can be convertedin the following manner.

Assuming that calcium is largely excessive in a solution containing 125mM of calcium and 0.75 mM of phosphoric acid and that all of phosphoricacid molecules are turned into CaP, the composition of CaP isCa₂(OH)(PO₄)₃, and therefore a CaP weight concentration is 126 μg/ml(Table 1). Further, assuming that all of PRG-PAA added are adsorbed onCaP, the composition ratio shown in Table 2 is obtained.

TABLE 1 Weight concentration in components of CaP particle solutionSample PRG-PAA CaP DNA Total No. (μg/ml) (μg/ml) (μg/ml) (μg/ml) 1 70126 16 212 2 140 126 11 277 3 210 126 7 343 4 280 126 5.3 411

TABLE 2 Component composition ratio of CaP particles Evaluation PRG-PAA/CaP/ DNA/ No. weight % weight % weight % 1 33 60 7.4 2 51 46 3.8 3 61 372 4 68 31 1.3

The eluents 1 and 2 described in the experiments described above wereused for the following purposes respectively.

Eluent 1: a calcium concentration of 125 mM in the eluent 1 is the sameas that of the CaP particle solution. The calcium concentration islargely excessive to that of phosphoric acid on this condition, and itis considered that the CaP particles are not dissolved. In this case,this eluent was used to determine a DNA-including amount in the CaPparticles prepared.

Eluent 2: a factor exerting an effect on the stability of the particleson a physiological condition is the concentrations of calcium andphosphoric acid contained in the solution. In this case, a solutioncontaining calcium and phosphoric acid each having a concentration closeto the physiological condition was used as the eluent to evaluate thestability of the particles on the physiological condition.

Example 5 Movement of DNA Included in Particles in Cell

CaP particles were prepared in the same manner as in Example 1. Providedthat DNA of 20 mer subjected to fluorescent labeling with Rhodamine wasused. The aqueous dispersion was left standing still at 25° C. for anight and then worked on a cell.

Experiment of Action on Cell

A cell strain HuH-7 (originating in human liver cancer, obtained fromRiken Gene Bank) was cultivated at 37° C. under 5% CO₂ atmosphere usinga culture medium prepared by adding 10% fetal calf serum to DMEM(Dulbecco's modified eagle medium; obtained from GIBCO BRL).

The aqueous dispersion was added to the cultured substance describedabove in a proportion of 1 to 9 of the CaP particle-containing aqueousdispersion to the culture medium in terms of a volume ratio to cultivatethe strain for 3 hours, and then the culture medium was removed,followed by washing the strain three times with PBS. This was observedunder a confocal laser microscope LSM510 (manufactured by Carl ZeissCo., Ltd.). Photographs in place of drawings showing the state of thetreated cells are attached as FIG. 6 to 8.

CaP/DNA was adsorbed on the surface of the cell on a condition on whicha precipitate was formed (*precipitate was observed in PEG-PAA 70 μg/mlbecause of a difference in a DNA chain length). On the other hand, itwas suggested from a granular fluorescent image that the CaP particleswere introduced into the cell by endocytosis.

Further, it was suggested that when adding (35 μg/mL, DNA 35 μg/mL)poly(methacrylic acid) (molecular weight 9500; obtained from AldrichChemical Company Inc.) in forming the particles, DNA is localized in thenucleus of the cell. DNA is distributed in the cytoplasma only withRhodamine, and therefore it is considered that DNA is not broken toexcess in this case.

Example 6 Particle Diameter of Composite Particles of DNA and CalciumPhosphate (No. 2)

It was shown in Example 2 that a particle diameter of the compositeparticles could be controlled by changing a concentration of thecopolymer, and it shall be shown in the present example that the aboveparticle diameter can further be controlled by changing a concentrationof phosphoric acid together with a concentration of the copolymer.

<Experiment>

(1) The following solution was prepared:

Solution A: DNA (16 mer: 70 μg/mL) 1/10 TE buffer (pH 7.6) Ca⁺² 250 mM(using CaCl₂) Solution B: PO₄ ³⁻ 1.5 mM, 3.0 mM or 6.0 mM (usingNa₂HPO₄) Hepes buffer 50 mM (pH 7.05) NaCl 140 mM PEG-PAA (PEG molecularweight: 12000, PAA polymerization degree: 24) 140 to 1400 μg/mL(2) The solution A was mixed with the solution B each in the sameamount, and the dispersion was left standing still at 37° C. for anight. Then, a particle diameter and a polydispersity of the particleswere determined by dynamic light scattering measurement.<Result>

As shown in FIG. 9( a), a particle diameter of the particles formed waschanged by the concentrations of PEG-PAA and phosphoric acid, and asshown in FIG. 9( b), the uniform particles having a polydispersity of0.1 or less and a particle diameter of 100 to 300 nm were obtained inany concentration. The marks □, ⋄ and ◯ in FIG. 9( a) and (b) eachcorrespond to the phosphoric acid concentrations of 0.75 mM, 1.5 mM and3.0 mM.

Example 7 Determination of Amount of DNA Introduced (or Included) intoParticles (No. 2)

Particles prepared according to Example 6 were quantitatively determinedfor an including amount of DNA in the same manner as described inExample 4.

<Result>

The measurement result of HPLC is shown in FIG. 10. It is observed fromthe drawing that an including amount of DNA tends to go up as aconcentration of phosphoric acid is increased. An inclusion rate of DNAreached 90% in 3.0 mM of phosphoric acid and 540 μg/mL of PEG-PAA.Comparing the results of the eluent 1 with those of the eluent 2, theinclusion rate was observed to be decreased in the eluent 2 in which acalcium concentration and a phosphoric acid concentration were close tothose of a human being. In the drawing, a void shows a result obtainedby using the eluent 1, and a solid mark shows a result obtained by usingthe eluent 2. Numerals in the drawing means the phosphoric acidconcentrations.

Example 8 Introduction of DNA-Including Particles Into Cell Experiment

1. Particle Preparing Condition

(1) The following solution was prepared:

Solution A: DNA (16 mer: 70 μg/mL) (5′ end was labelled with FITC) 1/10TE buffer (pH 7.6) Ca⁺² 250 mM Solution B: PO₄ ³⁻ 6.0 mM Hepes buffer 50mM (pH 7.05) NaCl 140 mM PEG-PAA (PEG molecular weight: 12000, PAApolymerization degree: 24) 1080 μg/mL(2) The solutions A and B were mixed in the same amount and leftstanding still at 37° C. for 24 hours.2. Evaluation for Introducing Cell

An HeLa cell (human cervix cancer cell) was put on a 24-well plate in acell density of 2×10⁴/well and cultivated in a DMEM culture medium(containing 10% FCS) for 24 hours. Then, 50 μL of the particles or a DNAsample was added to 450 μL of the culture medium (DMEM, 10% FCS). Afterprescribed time passed, the cell was peeled off by trypsinization anddispersed in 1 to 2 mL of PBS (cooled with ice until measurement). Thecell of about 2×10³ was analyzed by flow cytometry.

A gate was applied so that the untreated cell having the strongestfluorescent intensity was contained in a proportion of 1%. A fluorescentintensity of the untreated cell had an average value of 0.5.

<Result>

The results are shown in FIG. 11( a), (b) and (c). It can be found fromthe drawing that DNA alone is scarcely observed to be introduced on theexperimental condition described above. On the other hand, it has beenapparent that introduction of DNA into the cell is increased to a largeextent by allowing DNA to be incorporated in the particles and thatnucleic acid is introduced into almost 100% of the cells in 4 hours. Itcan be found as well from a result obtained by plotting an averagefluorescent intensity of the cell that introduction of DNA into the cellis promoted to a large extent as compared with DNA alone by making useof the particles.

FIG. 11( a) is a typical histogram (after 24 hours) of the untreatedcell and the cell treated with the CaP particles; (b) shows a change inan amount of introducing DNA into a cell versus time (shown by % of thecell present in a gate area); and (c) shows a change in an amount ofintroducing DNA into a cell versus time (shown by the averagefluorescent intensity).

Example 9 Toxicity Evaluation Test of Particles Experimental Method

1. Particle Preparing Conditions

(1) The following solution was prepared:

Solution A: DNA (16 mer: 70 μg/mL) 1/10 TE buffer (pH 7.6) Ca⁺² 250 mMSolution B: PO₄ ³⁻ 1.5 mM Hepes buffer 50 mM (pH 7.05) NaCl 140 mMPEG-PAA (PEG molecular weight: 12000, PAA polymerization degree: 24) 140to 560 μg/mL(2) The solutions A and B were mixed in the same amount and leftstanding still at 37° C. for 24 hours.2. Toxicity Test

An HeLa cell (human cervix cancer cell) was put on a 96-well plate in acell density of 5×10³/well and cultivated in a DMEM culture medium(containing 10% FCS) for 2 days. Then, 10 μL of a particle sample wasadded to 90 μL of the culture medium (DMEM, 10% FCS). After cultivatedfor 24 hours, the viable cell number was counted by MTT(3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide)assay.

The MTT assay was carried out in the following manner.

An MTT (5 mg/mL (PBS)) solution was added in an amount of 10 μL per wellof the 96-well plate and left standing still at 37° C. for 2 hours.Then, 20% SDS (DMF:water=1:1) was added in an amount of 100 μL per welland left standing still at 37° C. for 24 hours. The absorbance at 560 nmwas measured by a plate leader.

<Result>

The result is shown in FIG. 12. FIG. 12 shows percentage of a viablecell number to an untreated cell by a bar graph. According to thisgraph, it is apparent that the viable cell number is not decreased ascompared with the untreated cell within an experimental error range andthat the particles do not show a marked toxicity under the existingexperimental conditions.

Example 10 Transfection Experiment

Plasmid DNA (coded with a luciferase gene) was used as included nucleicacid in the following experiment to investigate an ability of theparticles for introducing a gene into a cell.

<Experimental Method>

1. Particle Preparation

(1) The following solution was prepared:

Solution A: Plasmid DNA (pGL3-Luc, 5 kbp) (26 to 50 μg/mL) 1/10 TEbuffer (pH 7.6) Ca⁺² 250 mM Solution B: PO₄ ³⁻ 1.5 mM Hepes buffer 50 mM(pH 7.05) NaCl 140 mM PEG-PAA (PEG molecular weight: 12000, PAApolymerization degree: 24) 100 to 200 μg/mL(2) The solutions A and B were mixed in the same amount and leftstanding still at 25° C. for 24 hours.2. Transfection Experiment

293T cell (human kidney) was put on a 6-well dish (coated with gelatin)so that 60% confluent was achieved and cultivated in a DMEM culturemedium (containing 10% FCS) for 24 hours. After removing the culturemedium, the cell was washed with PBS, and a new culture medium was addedthereto. Then, the sample was added in an amount of 100 μL per mL of theculture medium and cultivated for 6 hours. After exchanging the culturemedium, the cell was further cultivated for 24 hours. The luciferasegene-revealing amount was quantitatively determined by means of acommercial assay kit.

<Result>

The results are shown in FIGS. 13A and B. FIG. 13A shows a resultobtained by examining an influence of a PEG-PAA temperature and thepresence of a blood serum in cultivating for 6 hours to the revelationof a gene. The activity which was higher by 4 to 6 times as comparedwith that in 0 μg/mL was observed in 50 μg/mL of PEG-PAA. Further, it isapparent that the activity revealed is decreased as the PEG-PAAconcentration is increased to 75 and 100 μg/mL.

The expression amount is reduced on the condition of 50 and 75 μg/mL inthe presence of a blood serum as compared with in the absence thereof,but the activity which is higher than or equivalent to that in 0 μg/mLis obtained.

FIG. 13B shows a result obtained by examining an influence of theplasmid DNA concentration to the revelation of a gene.

In a PEG-PAA concentration of 0 μg/mL, a notable difference inrevelation caused by the plasmid DNA concentration was not observed. Onthe other hand, in 50 μg/mL, the revelation went up as the plasmid DNAconcentration was raised.

INDUSTRIAL APPLICABILITY

The organic-inorganic hybrid particle according to the present inventionis useful for introducing a biologically active substance (for example,DNA) into, for example, a cell and expressing it. Accordingly, thepresent invention is applicable in the medical industry and thepharmaceutical industry.

1. A composition comprising organic-inorganic hybrid particles having abiologically active substance carried thereon, wherein the particles areformed from a block copolymer having a structure represented by any oneof the following Formulas (II-a), (II-b), (III-a) and (III-b):

where in the respective formulas, A, L, T, m, x, y and z each have thefollowing independent meanings; A represents a hydrogen atom or asubstituted or unsubstituted alkyl group having up to 12 carbon atoms; Lrepresents a single bond, NH, CO or X(CH₂)_(p)Y, in which X representsOCO, OCONH, NHCO, NHCOO, NHCONH, CONH or COO; Y represents NH or CO; andp represents an integer of 1 to 6; T represents a hydrogen atom, ahydroxyl group or —ZR, in which Z represents a single bond, CO, O or NH,and R represents a substituted or unsubstituted hydrocarbon group havingup to 12 carbon atoms; m represents an integer of 4 to 2500; and x+y orz represents an integer of 5 to 300, a calcium ion (Ca⁺²), a phosphoricacid ion (PO₄ ³⁻) and the biologically active substance, and theparticles have an average particle diameter of 50 to 600 nm in anaqueous dispersion, and are substantially spherical, wherein the calciumion is present in the hybrid particles in an amount which is excessiveto the phosphoric acid ion as compared with an equivalent required forforming hydroxyapatite, and wherein the organic-inorganic hybridparticles comprise 30 to 70% by weight of the block copolymer, 25 to 65%by weight of hydroxyapatite and 0.1 to 15% by weight of the biologicallyactive substance each based on the whole weight of the particles.
 2. Thecomposition as described in claim 1, wherein m is 12 or more, and x+y orz is 50 or less.
 3. The composition as described in claim 1, wherein theorganic-inorganic hybrid particles have any diameter of an averageparticle diameter of 50 to 600 nm and a polydispersity of 0.1 or less inthe aqueous dispersion.
 4. The composition as described in claim 1,wherein the biologically active substance is selected from the groupconsisting of poly- or oligonucleotide and poly- or oligopeptide.
 5. Thecomposition as described in claim 1, wherein the composition comprisingthe organic-inorganic hybrid particles has the form of an aqueousdispersion.
 6. The composition as described in claim 1, wherein thecomposition comprising the organic-inorganic hybrid particles has afreeze-dried form.
 7. A method for preparing the composition asdescribed in claim 1, comprising: (A) a step of preparing a firstaqueous solution containing a biologically active substance, and acalcium ion, (B) a step of preparing independently a second aqueoussolution containing a block copolymer having a structure represented byany one of the following Formulas (II-a), (II-b), (III-a) and (III-b):

where in the respective formulas, A, L, T, m, x, y, and z each have theindependent meanings recited in claim 6; and a phosphoric acid ion and(C) a step of mixing the first aqueous solution with the second aqueoussolution to form hydroxyapatite, wherein the calcium ion is present inthe hybrid particles in an amount which is excessive to the phosphoricacid ion as compared with an equivalent required for forminghydroxyapatite, and wherein the organic-inorganic hybrid particlescomprise 30 to 70% by weight of the block copolymer, 25 to 65% by weightof hydroxyapatite and 0.1 to 15% by weight of the biologically activesubstance each based on the whole weight of the particles.
 8. The methodas described in claim 7, wherein a proportion of Ca⁺² to PO₄ ³⁻ is 50 to200:1 in terms of mole concentration.
 9. The method as described inclaim 7, wherein the biologically active substance is selected from thegroup consisting of poly- or oligonucleotide and poly- or oligopeptide.10. The composition as described in claim 1, wherein a proportion ofCa⁺² to PO₄ ³⁻ is 50 to 200:1 in terms of mole concentration.
 11. Amethod for producing a dispersion comprising organic-inorganic hybridtype particles having an average particle diameter of 50 to 600 nm,comprising a step of carrying out reaction using a calcium ion and aphosphoric acid ion in an aqueous solution in which a block copolymerrepresented by any one of the following Formulas (II-a), (II-b), (III-a)and (III-b) is present, wherein an excess calcium ion exceeding anequivalent required for forming hydroxyapatite is used:

in the respective formulas, the respective codes each have independentmeanings; A represents a hydrogen atom or a substituted or unsubstitutedalkyl group having up to 12 carbon atoms; L represents a single bond,NH, CO or X(CH₂)_(p)Y, in which X represents OCO, OCONH, NHCO, NHCOO,NHCONH, CONH or COO; Y represents NH or CO; and p represents an integerof 1 to 6; T represents a hydrogen atom, a hydroxyl group or —ZR, inwhich Z represents a single bond, CO, O or NH, and R represents asubstituted or unsubstituted hydrocarbon group having up to 12 carbonatoms; m represents an integer of 4 to 2500; and x+y or z represents aninteger of 5 to 300, provided that a carboxylate ion present can form acarboxyester residue in an amount of up to 50%, wherein the particlesare substantially spherical and comprise 30 to 70% by weight of theblock copolymer and 25 to 65% by weight of hydroxyapatite.